Methods of treating friedreich&#39;s ataxia using src inhibitors

ABSTRACT

In one aspect, the invention presents a method of treating Friedreich&#39;s ataxia (FRDA) by administering a therapeutically effective amount of an Src inhibitor.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of International Patent Application No. PCT/IB2015/059963 (filed Dec. 23, 2015), which claims the benefit of U.S. Provisional Patent Application 62/096,436 (filed on Dec. 23, 2014). Both applications are incorporated by reference in their entirety for all purposes.

REFERENCE TO SUBMISSION OF A SEQUENCE LISTING

This application includes a sequence listing as a text file named “SEQ_097459-1053552-000210US_ST25.txt,” created Jun. 22, 2017, and containing 817 bytes. The material contained in this text file is incorporated by reference in its entirety for all purposes.

BACKGROUND OF THE INVENTION

Friedreich's ataxia (FRDA) is an autosomal recessive disorder characterized by progressive degeneration of the central and peripheral nervous systems, cardiomyopathy, and increased incidence of diabetes mellitus. FRDA typically substantially impairs patient mobility and shortens lifespan. There is no current approved treatment for FRDA.

FRDA is caused by homozygous hyperexpansion of GAA triplets in intron 1 of the frataxin (FXN) gene on chromosome 9q13. Pastore, A.; Puccio, H. (2013), J. Neurochemistry, 126 Suppl 1, 43-52. Pathological GAA expansions severely reduce transcription of the FXN gene, resulting in low levels of frataxin expression. Frataxin is an extremely conserved mitochondrial protein synthesized as a cytosolic 210 amino acid precursor, which is then imported into mitochondria following a two-step proteolytic processing by a mitochondrial processing peptidase. Condo, I.; Ventura, N.; Malisan, F.; Rufini, A.; Tomassini, B.; Testi, R. (2007), Hum Mol Genet, 16, 1534-40; Schmucker, S.; Argentini, M.; Carelle-Calmels, N.; Martelli, A.; Puccio, H. (2008), Hum Mol Genet, 17, 3521-31.

Low levels of expression of frataxin are responsible for all clinical and morphological manifestations of FRDA. Evans-Galea, M. V.; Lockhart, P. J.; Galea, C. A.; Hannan, A. J.; Delatycki, M. B. (2014), Discovery Medicine, 17, 25-35. Low levels have also been correlated with disease severity. Parkinson, M. H.; Boesch, S.; Nachbauer, W.; Mariotti, C.; Giunti, P. (2013), 126 Suppl 1, 103-17.

Frataxin levels are not only crucial for cell survival, but also for stress handling responses. Condo, I.; Ventura, N.; Malisan, F.; Tomassini, B.; Testi, R. (2006), J Biol Chem, 281, 16750-56; Paupe, V.; Dassa, E. P.; Goncalves, S.; Auchere, F.; Lonn, M.; Holmgren, A. and Rustin, P. (2009) PLoS One, 4, e4253; Guccini, I.; Serio, D.; Condo, I.; Rufini, A.; Tomassini, B.; Mangiola, A.; Maira, G.; Anile, C.; Fina, D.; Pallone, F. et al. (2011), Cell Death Dis, 2, e123; Schiavi, A.; Torgovnick, A.; Kell, A.; Megalou, E.; Castelein, N.; Guccini, I.; Marzocchella, L.; Gelino, S.; Hansen, M.; Malisan, F. et al. (2013), Exp Gerontol, 48, 191-201; Hayashi, G.; Shen, Y.; Pedersen, T. L.; Newman, J. W.; Pook, M.; Cortopassi, G. (2014), Hum Mol Genet, 23, 6838-47.

In fact, frataxin deficiency in humans critically affects survival of large primary neurons of the dorsal root ganglia, cardiomyocytes, and pancreatic β-cells. As a consequence of dysregulated mitochondrial metabolism, frataxin-defective cells have reduced activity of iron sulfur cluster (ISC)-containing enzymes, a general imbalance in intracellular iron distribution, reduced ATP content, and increased sensitivity to oxidative stress, with increased ROS generation.

Because the frataxin coding sequence is intact in most FRDA patients, potential therapies aiming at enhancing frataxin levels are a useful target for development. Cf Hayashi, G.; Shen, Y.; Pedersen, T. L.; Newman, J. W.; Pook, M.; Cortopassi, G. (2014) Hum Mol Genet, 23, 6838-47; Herman, D.; Jenssen, K.; Burnett, R.; Soragni, E.; Perlman, S. L.; Gottesfeld, J. M. (2006) Nature Chemical Biology, 2, 551-58; Marmolino, D.; Acquaviva, F.; Pinelli, M.; Monticelli, A.; Castaldo, I.; Filla, A.; Cocozza, S. (2009), Cerebellum, 8, 98-103; Tomassini, B.; Arcuri, G.; Fortuni, S.; Sandi, C.; Ezzatizadeh, V.; Casali, C.; Condo, I.; Malisan, F.; Al-Mandawi, S.; Pook, M. et al. (2012) Hum Mol Genet, 21, 2855-61; Puccio, H.; Anheim, M.; Tranchant, C. (2014), I, 170, 355-65. Frataxin protein levels are controlled upon ubiquitination of target residue K147, a signal that targets frataxin to degradation by the proteasome. Barila, D.; Rufini, A.; Condo, I.; Ventura, N.; Dorey, K.; Superti-Furga, G.; Testi, R. (2003), Molecular and cellular biology, 23, 2790-99. This lysine represents a crucial site for frataxin stability since a frataxin mutant lacking K147 cannot be ubiquitinated and is more stable. A method of preventing ubiquitination on K147 could grant frataxin an increased stability and a prolonged half-life. Id.

Ubiquitination and phosphorylation are post-translational modifications (PTM) that often interact with each other dictating the fate of proteins. Hunter, T. (2007), Mol Cell, 28, 730-38. In addition, PTM have emerged as powerful modulators of metabolic pathways and are important regulators of mitochondrial functions. Hofer, A. and Wenz, T. (2014), Exp Gerontol, 56, 202-20. Hebert-Chatelain, E. (2013), Int J Biochem Cell Biol, 45,90-98. Moreover, Src tyrosine kinase family members such as Lyn, Fgr, Fyn and c-Src have also been reported to associate with mitochondria (Hunter, T. 2007). Src tyrosine kinases can be regulated by a variety of important mitochondrial signals such as ATP levels, redox state, which are dysregulated in FRDA. Hebert-Chatelain, E. (2013), Int J Biochem Cell Biol, 45, 90-98.

The present invention shows that blocking Src activity with Src inhibitors increases frataxin levels. Src inhibitors induce frataxin expression in cells derived from FRDA patients, though Src inhibitors failed to upregulate frataxin in human cells in which a frataxin-Y118F mutant was expressed. Therefore, Src inhibitors can promote frataxin accumulation in living cells, thus mitigating or alleviating the damaging effects of FRDA.

In one aspect, the present invention sets forth a method of preventing ubiquitination on K147 could grant frataxin an increased stability and a prolonged half-life. These and other advantages and solutions are presented by the invention disclosed herein.

BRIEF SUMMARY OF THE INVENTION

In one aspect, the invention provides a method of treating Friedreich's ataxia that includes administering to the subject a therapeutically effective amount of an Src inhibitor or a pharmaceutically acceptable salt.

In one aspect, the invention provides a method of modulating the phosphorylation of frataxin on Y118 by Src kinase. In one aspect, the invention provides a method of increasing frataxin levels in living cells, which may be in a subject suffering from FRDA or from low levels of frataxin. In one aspect, the method slows or otherwise palliates one or more clinical effects associated with FRDA or with low levels of frataxin.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1C. Src kinase phosphorylates frataxin. (FIG. 1A) HEK293 cells were transiently transfected with frataxin (FXN), and either constitutively active Src (Y527F) or its kinase inactive counterpart (Y527F-Kin⁻). Total protein extracts (TOT) were separated by SDS-Page and immunoblotted (WB) with specific antibodies against frataxin and tubulin (TUB) as loading control. Data are representative of ten independent experiments. (FIG. 1B) Total lysate of HEK293 transfected with frataxin and constitutively active Src (Y527F) was incubated for 50 minutes at 37° C. with buffer alone, CIP phosphatase (PPase) in presence or absence of phosphatase inhibitors (Inh) and analyzed after separation by SDS-Page by immunoblotting (WB) with specific antibody against frataxin (FXN). Data are representative of three independent experiments. (FIG. 1C) HEK293 cells were transiently transfected with frataxin, and either constitutively active Src (Y527F), its kinase inactive counterpart (Y527F-Kin) and constitutively active Abl (Abl-PP). Total protein extracts (TOT) or immunoprecipitated frataxin (IP) were separated by SDS-Page and immunoblotted (WB) with specific antibodies against frataxin (FXN), phosphorylated tyrosine (pY) and tubulin (TUB) as loading control. Data are representative of three independent experiments. The precursor (P), intermediate (I) and mature (M) frataxin forms are indicated. The arrows show the phosphorylated shifted precursor form.

FIGS. 2A-2B. Src kinase phosphorylates frataxin on residue Y118. (FIG. 2A) HEK293 cells were transiently transfected with wild type frataxin (FXN), or non-phosphorylable frataxin mutants Y175F, Y118F, Y123F, and either constitutively active Src (Y527F) or its kinase inactive counterpart (Y527F-Kin⁻). Total protein extracts (TOT) or immunoprecipitated frataxin (IP) were separated by SDS-Page and immunoblotted (WB) with specific antibodies against frataxin, phosphorylated tyrosine (pY) and tubulin (TUB) as loading control. Data are representative of three independent experiments. The precursor (P), intermediate (I) and mature (M) frataxin forms are indicated. (FIG. 2B) The figure shows the fragment ion spectrum of the phosphopeptide (96-123), with sequence ERLAEETLDSLAEFFEDLADKPpYTFEDY (SEQ ID NO:1) and precursor ion MH³⁺ 1146.7 m/z, which was obtained after in gel digestion of frataxin by chymotrypsin. The fragment ion series are indicated on the sequence: b and y ions, which are detected in the spectrum, are in bold dark. The upper panel shows an enlarged region of the spectrum (m/z range from 1200 to 1440): here, the distance from the peaks corresponding to b₂₂ to b₂₃ ions concides with a phosphotyrosine (pY) locating the phosphorylation on Y118.

FIGS. 3A-3B. Phosphorylation on Y118 promotes ubiquitination. (FIG. 3A) HEK293 cells were transiently transfected with wild type frataxin (FXN), or non-phosphorylable mutants Y118F, Y166F, Y175F, and hemaglutinin (HA)-tagged ubiquitin (HA-Ub). Total protein extracts (TOT) or immunoprecipitated ubiquitinated frataxin (IP α-HA) were separated by SDS-Page and immunoblotted (WB) with specific antibodies against frataxin (FXN), and tubulin (TUB) as loading control. Data are representative of five independent experiments. (FIG. 3B) The graph illustrates the relative ubiquitination level quantitated as the ratio between mono-ubiquitinated frataxin (Mono-Ub) level versus the frataxin precursor expression in the MG132-treated lanes. The precursor (P), intermediate (I) and mature (M) frataxin forms are indicated.

FIG. 4. Src inhibitors upregulate wild type frataxin but not the non-phosphorylable Y118F frataxin mutant. HEK293^(FXN) cells stably expressing single copy of wild type frataxin (WT) or non-phosphorylable Y118F frataxin mutant (Y118F) were treated for 24 h with 1, 3, and 10 μM of either Src inhibitor SU6656, PP2, dasatinib, bosutinib, saracatinib, or vehicle (−). Left panels: Frataxin (FXN) and tubulin expression (TUB) was analyzed by western blot. Data are representative of three independent experiments. The precursor (P), intermediate (I) and mature (M) frataxin forms are indicated. Right panels: Densitometric quantification of frataxin accumulation for the corresponding inhibitor. Frataxin expression was normalized with tubulin and frataxin expression in non-treated cells (−) set to one. Data represent the mean±1 S.E.M. from three different independent experiments performed for each inhibitor. P-values were calculated with the Student's t-test and were statistically significant (*P<0.05; **P<0.01) for each treatment compared to non-treated conditions.

FIG. 5. Src inhibitors promote endogenous frataxin accumulation in HEK293 cells. Human HEK293 cells were treated with 1, 3 and 10 μM of SU6656, PP2, and dasatinib Src inhibitor or vehicle (−) for 24 h. Frataxin (FXN) and tubulin expression (TUB) was analyzed by western blot. Data are representative of three independent experiments. The precursor (P), intermediate (I) and mature (M) frataxin forms are indicated. Right panels: densitometric quantification of frataxin accumulation. Frataxin expression was normalized with tubulin and frataxin expression in non-treated cells (NT) set to one. Data represent the mean±1 S.E.M. from three different independent experiments performed for each inhibitor in the left panels. P-values were calculated with Student's t-test and were statistically significant (*P<0.05; **P<0.01) for each treatment compared to non-treated conditions.

FIG. 6. Src inhibitors promote frataxin accumulation in frataxin-deficient cells. FRDA patient-derived B cells were treated with 10 μM of either Src inhibitor SU6656, PP2, dasatinib, or vehicle (−) for the time indicated. Left panels: mature frataxin (FXN) and tubulin expression (TUB) was analyzed by western blot. Data are representative of three independent experiments. Right panels: densitometric quantification of frataxin accumulation. Frataxin expression was normalized with tubulin and frataxin expression in non-treated cells (NT) set to one. Data represent the mean±1 S.E.M. from three different independent experiments performed for each inhibitor in the left panels. P-values were calculated with the Student's t-test and were statistically significant (*P<0.05; ** P<0.01) for each treatment compared to non-treated conditions.

FIGS. 7A-7B. FIG. 7A shows a western blot of the frataxin precursor (FXN) and tubulin expression (TUB) for cells treated with the Src inhibitor dasatinib (Dasa, 100 nM), the ubiquitin competing molecule F166 (1 uM), and a combination. Data are representative of four independent experiments. FIG. 7B show a densitometric quantification of frataxin accumulation. The frataxin expression was normalized with tubulin, and frataxin expression in non-treated cells (−) was set to 1. Data represent the mean±1 S.E.M. from four independent experiments. P-values were calculated with the Student's t-test and were statistically significant (*P<0.05).

DETAILED DESCRIPTION OF THE INVENTION Definitions

Unless otherwise stated, the following terms used in this application, including the specification and claims, have the definitions given below. It must be noted that, as used in the specification and the appended claims, the singular forms “a,” “an” and “the” include plural referents unless the context clearly dictates otherwise. Definition of standard chemistry terms may be found in reference works, including Carey and Sundberg (2007) “Advanced Organic Chemistry 5^(th) Ed.” Vols. A and B, Springer Science+Business Media LLC, New York. The practice of the present invention will employ, unless otherwise indicated, conventional methods of synthetic organic chemistry, X-ray crystallography, protein NMR, mass spectroscopy, protein chemistry, biochemistry, preparative and analytical methods of chromatography, recombinant DNA techniques and pharmacology, within the skill of the art.

Any terms not directly defined herein shall be understood to have the meanings commonly associated with them as understood within the art of the invention. Certain terms are discussed herein to provide additional guidance to the practitioner in describing the compositions, devices, methods and the like of aspects of the invention, and how to make or use them. It will be appreciated that the same thing can be said in more than one way.

Consequently, alternative language and synonyms can be used for any one or more of the terms discussed herein. No significance is to be placed upon whether or not a term is elaborated or discussed herein. Some synonyms or substitutable methods, materials and the like are provided. Recital of one or a few synonyms or equivalents does not exclude use of other synonyms or equivalents, unless it is explicitly stated. Use of examples, including examples of terms, is for illustrative purposes only and does not limit the scope and meaning of the aspects of the invention herein.

All references included herein are incorporated by reference in their entirety, except that in cases of contradiction (e.g., different, inconsistent definitions of the same term), the instant specification takes priority.

The term “about” as used herein to modify a numerical value indicates a defined range around that value. If “X” were the value, “about X” would generally indicate a value from 0.95X to 1.05X. Any reference to “about X” specifically indicates at least the values X, 0.95X, 0.96X, 0.97X, 0.98X, 0.99X, 1.01X, 1.02X, 1.03X, 1.04X, and 1.05X. Thus, “about X” is intended to teach and provide written description support for a claim limitation of, e.g., “0.98X.” When the quantity “X” only includes whole-integer values (e.g., “X amino acids”), “about X” indicates the values X, X−1, and X+1.

When the term “about” is applied to the beginning of a numerical range, it applies to both ends of the range. Thus, “from about 5 to 20%” is equivalent to “from about 5% to about 20%.” When “about” is applied to the first value of a set of values, it applies to all values in that set. Thus, “about 7, 9, or 11” is equivalent to “about 7, about 9, or about 11.” However, when the modifier “about” is applied to describe only the end of a range or only a later value in a set of values, it applies only to that value or that end of the range. Thus, the range “about 2 to 10” is the same as “about 2 to about 10,” but the range “2 to about 10” is not.

“Antibody” refers to a polypeptide comprising a framework region from an immunoglobulin gene or fragments thereof that specifically binds and recognizes an antigen. The recognized immunoglobulin genes include the kappa, lambda, alpha, gamma, delta, epsilon, and mu constant region genes, as well as the myriad immunoglobulin variable region genes. Light chains are classified as either kappa or lambda. Heavy chains are classified as gamma, mu, alpha, delta, or epsilon, which in turn define the immunoglobulin classes, IgG, IgM, IgA, IgD and IgE, respectively. Typically, the antigen-binding region of an antibody will be most critical in specificity and affinity of binding.

Antibodies can be described or specified in terms of the epitope(s) or portion(s) of a polypeptide of the present invention which are recognized or specifically bound by the antibody. The epitope(s) or polypeptide portion(s) can be specified as described herein, e.g., by N-terminal and C-terminal positions, by size in contiguous amino acid residues.

The linking term “comprising” or “comprise” as used herein is not closed. For example, “a composition comprising A” must include at least the component A, but it may also include one or more other components (e.g., B; B and C; B, C, and D; and the like).

As used herein, “or” should in general be construed non-exclusively. For example, an embodiment of “a composition comprising A or B” would typically present an aspect with a composition comprising both A and B. “Or” should, however, be construed to exclude those aspects presented that cannot be combined without contradiction (e.g., a composition pH that is between 9 and 10 or between 7 and 8).

“Pharmaceutically acceptable salt” refers to a salt of a compound of the invention which is made with counterions understood in the art to be generally acceptable for pharmaceutical uses and which possesses the desired pharmacological activity of the parent compound. Such salts include: (1) acid addition salts, formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like; or formed with organic acids such as acetic acid, propionic acid, hexanoic acid, cyclopentanepropionic acid, glycolic acid, pyruvic acid, lactic acid, malonic acid, succinic acid, malic acid, maleic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, 3-(4-hydroxybenzoyl) benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, 1,2-ethane-disulfonic acid, 2-hydroxyethanesulfonic acid, benzenesulfonic acid, 4-chlorobenzenesulfonic acid, 2-naphthalenesulfonic acid, 4-toluenesulfonic acid, camphorsulfonic acid, 4-methylbicyclo[2.2.2]-oct-2-ene-1-carboxylic acid, glucoheptonic acid, 3-phenylpropionic acid, trimethylacetic acid, tertiary butylacetic acid, lauryl sulfuric acid, gluconic acid, glutamic acid, hydroxynaphthoic acid, salicylic acid, stearic acid, muconic acid and the like; or (2) salts formed when an acidic proton present in the parent compound is replaced by a metal ion, e.g., an alkali metal ion, an alkaline earth ion, or an aluminum ion; or coordinates with an organic base such as ethanolamine, diethanolamine, triethanolamine, N-methylglucamine, morpholine, piperidine, dimethylamine, diethylamine and the like. Also included are salts of amino acids such as arginates and the like, and salts of organic acids like glucurmic or galactunoric acids and the like (see, e.g., Berge et al. (1977) J Pharm. Sci., 66, 1-19).

“Specific binding” refers to the ability of two molecules to bind to each other in preference to binding to other molecules in the environment. Typically, “specific binding” discriminates over adventitious binding in a reaction by at least two-fold, more typically by at least 10-fold, often at least 100-fold. Typically, the affinity or avidity of a specific binding reaction, as quantified by a dissociation constant, is about 10⁻⁷ M or stronger (e.g., about 10⁻⁸ M, 10⁻⁹M or even stronger).

In the Summary of the Invention above, Detailed Description, and the claims below, reference is made to particular features and aspects of the invention, including method steps. The disclosure of the invention in this specification includes all possible combinations of such particular features within the embodiments of the invention disclosed, at least to the extent that such combinations are non-contradictory. For example, if the Detailed Description presents an aspect with embodiments A, B, and C, it is understood that this discloses combined, more specific embodiments that include both aspects A and B, both aspects B and C, and both aspects A and C, as well as an embodiment with the features of A, B, and C.

Methods

In one aspect, the invention presents a method of treating Friedreich's ataxia in a subject in need thereof, including administering to the subject a therapeutically effective amount of a Src inhibitor or a pharmaceutically acceptable salt thereof.

In one aspect, the subject is a mammal. In one aspect, the mammal is a human.

In one aspect, the invention presents a method as set forth herein, wherein the Src inhibitor inhibits Y118 phosphorylation of frataxin.

In one aspect, the invention presents a method as set forth herein, wherein the Src inhibitor is a SrcY527F antagonist.

In one aspect, the invention presents a method as set forth herein, wherein the Src inhibitor increases endogenous frataxin levels by at least 5% over an inactive control. In one aspect, the Src inhibitor increases endogenous frataxin levels by at least 10% over an inactive control (e.g., 10%, 15%, or 20%). In one aspect, the Src inhibitor increases endogenous frataxin levels by at least 25% over an inactive control (e.g., 25%, 30%, 35%, 40%, or 45%).

In one aspect, the Src inhibitor increases endogenous frataxin levels by at least 50% over an inactive control (e.g., 50%, 55%, 60%, 65%, or 70%). In one aspect, the Src inhibitor increases endogenous frataxin levels by at least 75% over an inactive control (e.g., 75%, 80%, 85%, 90%, or 95%). In one aspect, the Src inhibitor increases endogenous frataxin levels by at least 100% over an inactive control (e.g., 100%, 105%, 110%, 115%, or 120%).

In one aspect, the Src inhibitor increases endogenous frataxin levels by at least 125% over an inactive control (e.g., 125%, 130% 135%, 140%, 145%, 150%, 155%, 160% 165%, 170%, 175%, 180%, 185%, 190%, 200%, 225%, or 250%).

In one aspect, the invention presents a method as set forth herein, wherein the Src inhibitor is selected from the group consisting of saracatinib (AZD-530), dasatinib, bafetinib, KX01, XL228, DCC-2036, ponatinib (AP24534), and TG100435. In one aspect, the Src inhibitor is AZD-530. In one aspect, the Src inhibitor is dasatinib. In one aspect, the Src inhibitor is bafetinib. In one aspect, the Src inhibitor is KX01 (a.k.a. KX2-391). In one aspect, the Src inhibitor is XL228. In one aspect, the Src inhibitor is DCC-2036. In one aspect, the Src inhibitor is ponatinib. In one aspect, the Src inhibitor is TG100435.

In one aspect, the invention presents a method as set forth herein, wherein the Src inhibitor is selected from the group consisting of SU6656, PP2, dasatinib, bafetinib, saracatinib, XL999, KX01, XL228, and bosutinib. In one aspect, the Src inhibitor is SU6656. In one aspect, the Src inhibitor is PP2. In one aspect, the Src inhibitor is XL999. In one aspect, the Src inhibitor is KX01 (a.k.a. KX2-391). In one aspect, the Src inhibitor is XL228. In one aspect, the Src inhibitor is bosutinib.

In one aspect, the invention presents a method as set forth herein, wherein the Src inhibitor is administered at a daily dose of from about 2 to 25 mg of compound per kg of the subject's body weight (mg/kg) (e.g., 1.8, 1.9, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28). In one aspect, the Src inhibitor is administered at a daily dose of from about 2 to 20 mg of compound per kg of the subject's body weight (mg/kg). In one aspect, the Src inhibitor is administered at a daily dose of from about 1 to 15 mg of compound per kg of the subject's body weight (mg/kg). In one aspect, the Src inhibitor is administered at a daily dose of from about 1 to 10 mg of compound per kg of the subject's body weight (mg/kg) (e.g., 0.9, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, or 11). In one aspect, the Src inhibitor is administered at a daily dose of from about 500 to 5000 mcg of compound per kg of the subject's body weight (mcg/kg). In one aspect, the Src inhibitor is administered at a dose of from about 100 to 1000 mcg of compound per kg of the subject's body weight (mcg/kg). In one aspect, the Src inhibitor is administered at a daily dose of from about 50 to 500 mcg of compound per kg of the subject's body weight (mcg/kg). In one aspect, the Src inhibitor is administered at a daily dose of from about 25 to 250 mcg of compound per kg of the subject's body weight (mcg/kg). In one aspect, the Src inhibitor is administered at a daily dose of from about 10 to 100 mcg of compound per kg of the subject's body weight (mcg/kg). In one aspect, the Src inhibitor is administered at a daily dose of from about 1 to 50 mcg of compound per kg of the subject's body weight (mcg/kg). In one aspect, the Src inhibitor is administered at a daily dose of from about 1 to 25 mg of compound per kg of the subject's body weight (mcg/kg). In one aspect, the Src inhibitor is administered at a daily dose of from about 1 to 10 mcg of compound per kg of the subject's body weight (mcg/kg).

In one aspect, the invention presents a method as set forth herein, wherein the Src inhibitor is administered by controlled release. In one aspect, the Src inhibitor is administered topically. In one aspect, the Src inhibitor is administered intraveneously. In one aspect, the Src inhibitor is administered orally.

In one aspect, the invention presents a method as set forth herein, wherein the Src inhibitor is administered once weekly. In one aspect, the Src inhibitor is administered twice weekly. In one aspect, the Src inhibitor is administered three time weekly. In one aspect, the Src inhibitor is administered once daily. In one aspect, the Src inhibitor is administered twice daily. In one aspect, the Src inhibitor is administered three times daily.

In one aspect, the invention presents a method as set forth herein, wherein the method of treating Friedreich's ataxia comprises inhibiting ubiquitination of frataxin.

In one aspect, the invention presents a method of inhibiting ubiquitination of frataxin in a subject comprising administering to a subject a therapeutically effective amount of a Src inhibitor or a pharmaceutically acceptable salt thereof. In one aspect, the method is as otherwise set forth herein.

In one aspect, the invention presents a method as set forth herein, further comprising administering to the subject one or more agents selected from the group consisting of a ubiquitination inhibitor, an antioxidant, and a siderophore.

In one aspect, the invention presents a method as set forth herein, further comprising administering to the subject one or more agents selected from the group consisting of an interferon and a ubiquitin-competing molecule. In one aspect, the agent is an interferon. In one aspect, the interferon is gamma interferon, for example, using a method as set forth in U.S. Pat. No. 8,815,230.

In one aspect, the invention presents the use of an Src inhibitor in a method of treating Friedreich's ataxia in a subject in need thereof, comprising the method as set forth herein.

EXAMPLES Example 1 General Methods

Cell Cultures and Transfections.

Human embryonic kidney HEK-293 cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS). HEK-293 cells were transfected with the calcium/phosphate precipitation method, using 20 μg of total DNA (5 or 10 μg of pIRES2frataxin, 10 μg of Tyrosine mutated non-phosphorylable frataxin mutants, 10 μg of Src or Abl constructs and 10 μg of HA-Ub, or the corresponding empty vectors) on 10 cm dishes.

HEK-293 Flp-In cells (Life Technologies) are HEK-293 variants allowing the stable and isogenic integration and expression of a transfected gene. The HEK-293 clone stably expressing frataxin¹⁻²¹⁰ (Condo et al. 2007) and the clone stably expressing frataxin^(V118F) were generated by mutagenesis as described below. HEK-293 Flp-In cells were maintained in DMEM supplemented with 10% FBS.

Immortalized GM16214 lymphoblasts, from a clinically affected FRDA patient were were obtained from NIGMS Human Genetic Cell Repository, Coriell Institute for Medical Research (Camden, N.J., USA) and cultured in RPMI 1640 supplemented with 15% fetal bovine serum.

DNA Constructs.

The construct pIRES2-frataxin¹⁻²¹⁰ contains human frataxin cDNA cloned into the bicistronic expression vector pIRES2-EGFP (BD Clontech) and was previously generated in this laboratory. Condo, I.; Ventura, N.; Malisan, F.; Tomassini, B.; Testi, R. (2006), J Biol Chem, 281, 16750-56. All the tyrosine mutant constructs were generated using the Quick-Change site-directed mutagenesis kit (Stratagene) with specific primers using pIRES2-frataxin¹⁻²¹⁰ as template. The HA-Ub construct was generated by M. Treier. Treier, M.; Staszewski, L. M.; Bohmann, D. (1994) Cell, 78, 787-98. Constitutively active Src (pSGTSrcY527F), its inactive kinase counterpart (pSGTSrcY527F-kin-) and constitutively active Abl (pSGTAbl-PP) have been previously described. Barila, D.; Rufini, A.; Condo, I.; Ventura, N.; Dorey, K.; Superti-Furga, G.; Testi, R. (2003), Molecular and cellular biology, 23, 2790-99. All the constructs generated were verified by DNA sequencing.

Dephosphorylation Assay.

CIP dephosphorylation assay kit (New England BioLabs® Inc.) was used to release phosphate groups from residues of tyrosine. 50 units of CIP (Alkaline Phosphatase, Calf Intestinal) were added to total cell extracts resuspended in NE3 buffer pH 7.9 (1 M NaCl, 0.5 M Tris-HCl, 100 mM MgCl₂, 10 mM dithiothreitol), and incubated for 60 minutes at 37° C. Sodium orthovanadate 10mM and EDTA 50 mM were used to inhibit CIP activity.

Immunoprecipitation and Western Blot.

Total cell extracts were prepared in ice-cold modified RIPA buffer (10 mM sodium phosphate pH 7.2, 150 mM NaCl, 1% Na deoxycholate, 0.1% SDS, 1% Nonidet P-40, 2 mM EDTA) or IP buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1% Nonidet P-40, 5 mM EDTA, 5 mM EGTA) supplemented with complete protease inhibitor cocktail (Roche Diagnostics, Milan, Italy), sodium orthovanadate 1 mM and NaF 25 mM to inhibit phosphatases.

For in vivo detection of ubiquitin conjugates, 100 μM MG132, 50 ng/ml Ub-aldehyde and N-ethylmaleimide 2 mM (NEM; Sigma-Aldrich) were added to the lysis buffer. Cell lysates (100 μg) were resolved by SDS-PAGE and analyzed by immunoblot with specific mAb anti-frataxin clone 1G2 and STR-23 (Immunological Sciences, Rome, Italy) mAb anti-tubulin (Sigma), mAb anti-actin (Sigma), mAb anti-phosphotyrosine (Millipore), pAb anti-phospho-Src (Life Technologies), mAb anti-GFP (Takara), secondary antibody horseradish peroxidase-conjugated goat anti-mouse (Pierce), secondary antibody horseradish peroxidase-conjugated mouse anti-rabbit (Pierce), secondary antibody horseradish peroxidase-conjugated goat anti-Fc anti-mouse (Thermo Scientific) using ECL system detection (GE Healthcare Europe GmbH, Milan, Italy).

For immunoprecipitation, 5 mg of total protein extract prepared as above were incubated for 1-2 h at 4° C. with specific antibodies, previously conjugated to protein G-Sepharose (GE Healthcare). Immunocomplexes were then resolved and analysed by SDS-PAGE.

Densitometric analyses were performed using ImageLab software (Biorad).

MS/MS identification of nitrated or phosphorylated residues in Frataxin. Human Recombinant Frataxin protein (GenScript Corp., New Jersey, USA) was previous treated with Src Kinase Assay (Millipore) and then separated on 1D-gel NuPAGE 4-12% (Novex, Invitrogen) run in morpholinepropanesulfoninic acid (MOPS) buffer and stained with the Colloidal Blue Staining kit (Invitrogen).

The stained bands were cut from the gel and destained with a solution containing 50 mM ammonium bicarbonate/acetonitrile (1:1 v/v) (CH3CN, Merck Darmstadt, Germany). Protein bands were subsequently subjected to cysteine reduction by 10 mM DTT for 1 h at 56° C. and alkylation by 50 mM iodoacetamide for 45min at RT in the dark, then dried by acetonitrile treatment and finally in the speedvac apparatus.

In gel digestion was performed by incubating gel particles with a solution containing 12.5 ng/ml chymotrypsin (Promega, Madison, Wis., USA) in 25 mM ammonium bicarbonate at 37° C. overnight under stirring.

To recognize phosphorylated residues, peptide mixture was analyzed by nanoflow-reversed-phase liquid chromatography tandem mass spectrometry (RP-LC-MS/MS) using an HPLC Ultimate 3000 (DIONEX, Sunnyvale, Calif. U.S.A.) connected on line with a linear Ion Trap (LTQ, ThermoElectron, San Jose, Calif.). Peptides were desalted in a trap-column (AcclaimPepMap100 C18, LC Packings, DIONEX) and then separated in a reverse phase column, a 10-cm-long fused-silica capillary (SilicaTipsFS 360-75-8, New Objective, Woburn, Mass., USA), slurry-packed in-house with 5 μm, 200 Å pore size C18 resin (Michrom BioResources, Calif.). Peptides were eluted using a linear gradient from 96% aqueous phase (H₂O with 5% ACN, 0.1% formic acid) to 60% organic buffer (ACN with 5% H₂O with 0.1% formic acid) in 30 min, at 300 nl/min flow rate. Analyses were performed in positive ion mode and the HV Potential was set up around 1.5-1.8 kV. The LTQ mass spectrometer operated in a data-dependent mode in which each full MS scan was followed by five MS/MS scans where the five most abundant molecular ions were dynamically selected and fragmented by collision-induced dissociation (CID) using a normalized collision energy of 35%. Tandem mass spectra were matched against Swiss-Prot protein database and through SEQUEST algorithm (Yates et al., 1995) incorporated in Bioworks software (version3.3, Thermo Electron) using no enzyme constrain, static cysteine alkylation by iodoacetamide, variable modification by oxidation on methionine and phosphorylation on tyrosine residues (Δm: +80 Da). A peptide has been considered legitimately identified when it achieved cross correlation scores of 1.8 for [M+1]¹⁺, 2.5 for [M+2H]²⁺, 3 for [M+3H]³⁺, and a probability cut-off for randomized identification of p<0.001.

Reagents and Treatments.

The proteasome inhibitor MG132 (Sigma-Aldrich) and the deubiquitinase inhibitor Ub-aldehyde (Biomol) were added to the cell culture at the final concentration of 10 μM and 50 ng/mL respectively. The Src inhibitors PP2, SU6656 (EMD Millipore), bosutinib, dasatinib and saracatinib (Selleckchem) were added to cells at the minimum concentration of 10 nM to a maximum concentration of 10 μM. For treatment longer than 24 h, inhibitors were added every 2 days, and each time lymphoblasts were centrifuged and resuspended at 400.000 cells/ml with the indicated concentration of inhibitor.

Example 2 Src Kinase Triggers Frataxin Phosphorylation

To assess whether frataxin could be a substrate for non-receptor tyrosine kinases, frataxin was transiently transfected in human embryo kidney (HEK) 293 cells, together with several constructs encoding different forms of Src and Abl kinases. The constitutively active Src, SrcY527F, but not its inactive kinase counterpart, SrcY527FKin-, caused retarded frataxin precursor electrophoretic shift migration as shown by immunoblotting (FIG. 1A).

The mutants were analyzed in cotransfection assays with constitutively active SrcY527F and its kinase inactive counterpart SrcY527FKin-. HEK293 cells were transiently transfected with frataxin (FXN), and either constitutively active Src (Y527F) or its kinase inactive counterpart (Y527F-Kin) (FIG. 1A). The total protein extracts (TOT) were separated by SDS-Page and immunoblotted (WB) with specific antibodies against frataxin and tubulin (TUB) as loading control. The data shown are representative of ten independent experiments.

A total lysate of HEK293 transfected with frataxin and constitutively active Src (Y527F) was incubated for 50 minutes at 37° C. with buffer alone, and with CIP phosphatase (PPase) in the presence or absence of phosphatase inhibitors (Inh). It was analyzed after separation by SDS-Page by immunoblotting (WB) with specific antibody against frataxin (FXN) (FIG. 1B). The data shown are representative of three independent experiments.

HEK293 cells were transiently transfected with frataxin, and either constitutively active Src (Y527F), its kinase inactive counterpart (Y527F-Kin) and constitutively active Abl (Abl-PP). Total protein extracts (TOT) or immunoprecipitated frataxin (IP) were separated by SDS-Page and immunoblotted (WB) with specific antibodies against frataxin (FXN), phosphorylated tyrosine (pY) and tubulin (TUB) as loading controls (FIG. 1C). The data shown are representative of three independent experiments.

To address whether this shift migration was due to precursor phosphorylation, phosphatase assay on total lysates was performed. Following phosphatase treatment, the shifted form disappeared, indicating that the frataxin precursor is indeed phosphorylated in the cells (FIG. 1B).

To further validate frataxin precursor phosphorylation, immunoprecipitation experiments were performed (FIG. 1C). Interestingly, the constitutively active mutant of c-Abl, Abl-PP, kinase activity of which was indeed controlled (data not shown) could not phosphorylate frataxin precursor suggesting that the frataxin precursor is specifically phosphorylated by Src.

Example 3 Src Kinase Phosphorylates Frataxin on Residue Y118

To identify Src-induced tyrosine phosphorylated site(s) on frataxin, single non-phosphorylable mutants of the eight tyrosine residues (Y74, Y95, Y118, Y123, Y143, Y166, Y175, and Y205) were generated converting tyrosines into phenylalanine residues.

Interestingly, the mutation of residues Y95, Y118, and Y123 induced an electrophoretic shift migration of all the frataxin forms (FIG. 2A). Though mutations are conservative, the shift migration may be due to charge modifications since the α-helix 1 (D91 to A114) is an acidic residue-rich region and the loop 1 (D115 to Y123) is important for proper protein folding and stability. Roman, E. A.; Faraj, S. E.; Gallo, M.; Salvay, A. G.; Ferreiro, D. U.; Santos, J. (2012), PLoS One, 7, e45743.

FIG. 2A shows three representative mutants, out of the eight analyzed, while the associated table summarizes the results for all mutants. Only mutation of Y118 abrogated tyrosine phosphorylation of frataxin precursor, indicating that Y118 is the main Src phosphorylation site.

To further confirm that Y118 was the main phosphorylation site, mass spectrometry was performed (FIG. 2B). In vitro phosphorylation reaction using recombinant frataxin¹⁻²¹⁰ and recombinant Src was performed, recovered on SDS-PAGE gel after visualization by Coomassie staining, digested with chymotrypsin, and analyzed by MALDI-TOF mass spectrometry. One phosphopeptide was isolated, corresponding to amino acids 96-123, identifying the phosphorylated residue as Y118.

Example 4 Non-Phosphorylable Y118F Frataxin Mutant is Less Ubiquitinated

To evaluate the impact of Y118 on frataxin ubiquitination, HEK-293 cells were transfected with wild type frataxin or non-phosphorylable frataxin mutants Y118F, Y116F, and Y175F together with hemaglutinin (HA)-tagged ubiquitin (HA-Ub) in the absence or presence of proteasome inhibitor MG132. Using a previously reported procedure (Rufini et al. 2011), frataxin ubiquitination status was evaluated by anti-frataxin monoclonal antibodies on total lysates or after immunoprecipitation of ubiquitinated forms with anti-HA antibody only in the presence of MG132. Frataxin monoubiquitinated forms can be detected as slower migrating bands above the frataxin precursor.

FIG. 3 illustrates that accumulation of ubiquitinated frataxin forms was reduced when non-phosphorylable Y118F mutant, but not other non-phosphorylable mutants such as Y116F and Y175F, was transfected. Relative ubiquitination level was quantitated as the ratio between mono-ubiquitinated frataxin level versus the frataxin precursor expression in the MG132-treated lanes. Non-phosphorylable Y118F frataxin mutant is >60% less ubiquitinated compared to wild type frataxin, thus suggesting that phosphorylation on Y118 promotes (or is required for) ubiquitination.

Example 5 Src Inhibitors Increase Wild-Type Frataxin Expression, but not Non-phosphorylable Y118F Frataxin Mutant

Because phosphorylation on Y118 promotes ubiquitination and that preventing ubiquitin-dependent degradation increases frataxin levels (Rufini et al. 2011), the effects of frataxin phosphorylation by Src kinase were tested for enhancement of frataxin expression. Different Src inhibitors such as PP2, SU6656, saracatinib, bosutinib and dasatinib were used to treat HEK293^(FXN) cells, stably expressing a single copy of wild-type frataxin or the non-phosphorylable Y118F frataxin mutant.

As illustrated in FIG. 4, all the Src inhibitors can promote frataxin accumulation within 24 h in a dose-dependent manner, although with different efficacy. Dasatinib appeared to be the most efficient Src inhibitor tested, being still active in the nanomolar range of concentrations (data not shown). Interestingly, frataxin accumulation was observed for all the different frataxin forms such as precursor, intermediate and mature forms. All the Src inhibitors tested could increase frataxin levels in cells overexpressing wild-type frataxin, but not in cells overexpressing the non-phosphorylable Y118F frataxin mutant (FIG. 4), suggesting that they indeed act by inhibiting phosphorylation of Y118. In addition, these inhibitors are also effective on the endogenous frataxin as shown in HEK293 cells (FIG. 5).

Example 6 Src Inhibitors Promote Frataxin Accumulation in Frataxin-Deficient Cells

Since blocking Src activity with tyrosine kinase inhibitors increases frataxin levels in human cells, their efficacy was tested on frataxin-deficient cells, such as FDRA patient-derived B cells (GM16214). Among the different Src inhibitors, SU6656, PP2 and dasatinib seemed to be best tolerated. FRDA lymphoblasts were exposed to these inhibitors for different time periods. As illustrated in FIG. 6, the upregulation of frataxin could be detected as early as 24 h of treatment, and was further accumulated within 72 h, particularly with SU6656 and dasatinib.

This study showed that frataxin is phosphorylated by Src kinase on Y118, promoting its ubiquitination and degradation. Moreover, Src inhibitors increase frataxin expression in human cells. Accordingly, Src inhibitors failed to upregulate frataxin in human cells in which a frataxin Y118F mutant was expressed.

More importantly, Src inhibitors were effective in FRDA cells in promoting frataxin accumulation. The results showed the existence of a crosstalk between frataxin phosphorylation and ubiquitination controlling frataxin levels and provide the rationale for a new therapeutic strategy using Src inhibitors to increase frataxin levels in FRDA.

Example 7 Efficacy of Src Inhibitors In Vivo

The efficacy of dasatinib (TD Sprycel) and bosutinib (TD Bosulif) in the YG8R mouse model is investigated. These mice are engineered to represent a good proxy model for the progression and manifestation of FRDA (Anjomani Virmouni et al., PLOS One, 2014, http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0089488), as a recent study allowed the detection of neurological improvements as well as frataxin increases after a prolonged pharmacological treatment in this model. Tomassini et al. (2014), Hum. Mol. Genet. 21, 2855-61. Here, 8-week-old YG8R mice are treated for 14 weeks with the different Src inhibitors.

Dasatinib is delivered orally as a solution in citric acid buffer, with a regimen that has shown no toxicity in the nude mice. Lombardo et al. (2004), J. Med. Chem., 47, 6658-61. A daily dose of 50 mcg/kg, administered on a 5 days on and 2 days off schedule, will be tested.

Bosutinib is delivered orally at a daily dose of 100 mcg/kg according to a previously described therapeutic regimen in the nude mouse. Goias et al. (2005), Cancer Res., 65, 5358-64.

Treated mice, as well as vehicle treated mice, are subjected to a number of behavioural tests at regular intervals in time. These include body weight analysis, rotarod, beam-walk, hang wire, grip strength, and footprint tests. Moreover, at the end of the treatment, mice are sacrificed, and the amount of frataxin within the dorsal root ganglia neurons is quantitated by SDS-PAGE and Western blot analysis. These results clarify whether selected src inhibitors treatment has an impact on the progression of the disease in the mouse and whether this is associated with an increase of frataxin in critical neuronal compartments.

Example 8 Efficacy of Src Inhibitors with Ubiquitin Competing Molecules

Src inhibitors and ubiquitin competing molecules (Rufini et al. 2011; International Application No. PCT/IB2015/057318) can enhance each other's ability to promote frataxin precursor accumulation, as they impinge on the same molecular pathway. The Src inhibitor dasatinib further enhances the ability of the ubiquitin competing molecule F166 to accumulate frataxin precursor in living cells (FIG. 7).

HEK293^(FXN) cells stably expressing a single copy of wild-type frataxin were treated for 24 h with Src inhibitor dasatinib (Dasa, 100 nM), the ubiquitin competing molecule F166 (1 uM) or a combination thereof. The frataxin precursor (FXN) and tubulin expression (TUB) were analyzed by western blot (FIG. 7A). The data shown are representative of four independent experiments.

The amount of frataxin accumulation was quantified by densitometry (FIG. 7B). Frataxin expression was normalized with tubulin, and frataxin expression in non-treated cells (−) was set to 1. The data shown represent the mean±1 S.E.M. from four independent experiments. P-values were calculated with the Student's t-test and were statistically significant (*P<0.05).

This result provides proof of concept that the two classes of molecules can be used in combination in order to enhance therapeutic effects and/or to lower their pharmacological dosages, so to limit side-effects.

OTHER REFERENCES

Rufini A.; Cavallo F.; Condo I.; Fortuni S.; De Martino G.; Incani O.; Di Venere A.; Benini M.; Massaro D. S.; Arcuri G.; Serio D.; Malisan F.; Testi R. Highly specific ubiquitin-competing molecules effectively promote frataxin accumulation and partially rescue the aconitase defect in Friedreich ataxia cells. Neurobiol Dis. 2015, 75, 91-99. 

1. A method of treating Friedreich's ataxia in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a Src inhibitor or a pharmaceutically acceptable salt thereof.
 2. The method of claim 1, wherein the subject is a human.
 3. (canceled)
 4. The method of claim 1, wherein the Src inhibitor inhibits Y118 phosphorylation of frataxin.
 5. The method of claim 1, wherein the Src inhibitor is a SrcY527F antagonist.
 6. (canceled)
 7. The method of claim 1, wherein the Src inhibitor increases endogenous frataxin levels by at least 25% over an inactive control.
 8. The method of claim 1, wherein the Src inhibitor increases endogenous frataxin levels by at least 50% over an inactive control.
 9. (canceled)
 10. The method of claim 1, wherein the Src inhibitor increases endogenous frataxin levels by at least 100% over an inactive control.
 11. The method of claim 1, wherein the Src inhibitor is selected from the group consisting of saracatinib (AZD-530), dasatinib, bafetinib, KX01, XL228, DCC-2036, ponatinib (AP24534), and TG100435.
 12. The method of claim 1, wherein the Src inhibitor is selected from the group consisting of SU6656, PP2, dasatinib, bafetinib, saracatinib, XL999, KX01, XL228, and bosutinib.
 13. The method of claim 12, wherein the Src inhibitor is selected from the group consisting of SU6656, PP2, dasatinib, and bosutinib.
 14. The method of claim 12, wherein the Src inhibitor is dasatinib.
 15. The method of claim 12, wherein the Src inhibitor is bosutinib.
 16. (canceled)
 17. (canceled)
 18. (canceled)
 19. (canceled)
 20. (canceled)
 21. (canceled)
 22. (canceled)
 23. (canceled)
 24. (canceled)
 25. (canceled)
 26. (Canceled)
 27. (Canceled)
 28. The method of claim 1, wherein the Src inhibitor is administered by controlled release.
 29. The method of claim 1, wherein the Src inhibitor is administered topically.
 30. The method of claim 1, wherein the Src inhibitor is administered intraveneously.
 31. The method of claim 1, wherein the Src inhibitor is administered orally.
 32. (canceled)
 33. (canceled)
 34. (canceled)
 35. (canceled)
 36. (canceled)
 37. (canceled)
 38. (canceled)
 39. A method of inhibiting ubiquitination of frataxin in a subject comprising administering to a subject a therapeutically effective amount of a Src inhibitor or a pharmaceutically acceptable salt thereof.
 40. The method of claim 1, further comprising administering to the subject one or more agents selected from the group consisting of a ubiquitination inhibitor, an antioxidant, and a siderophore.
 41. The method of claim 1, further comprising administering to the subject one or more agents selected from the group consisting of an interferon and a ubiquitin-competing molecule.
 42. The method of claim 41, wherein the agent is an interferon.
 43. The method of claim 42, wherein the interferon is gamma interferon.
 44. (canceled) 